Cellular differentiation is associated with transcriptional induction of distinct sets of cell-type-specific genes whose expression is required for organ function. Deciphering the mechanisms that control cell-type-selective transcription is critical to understanding cellular differentiation. Studies of liver-specific DNA regulatory regions suggest that selectivity of hepatocyte-specific gene transcription relies on multiple DNA sequences that recruit binding and combinatorial interactions of distinct families of hepatocyte nuclear factors (HNF) to provide synergistic transcriptional activation. However, the mechanisms by which these liver transcription factors mediate synergistic transcriptional activation have not yet been clarified. We demonstrated that interactions between the Forkhead Box (Fox) A2 (HNF-3beta) and HNF-6 DNA binding domains result in stimulation of FoxA2 transcriptional activity through the recruitment of the p300/CBP coactivator proteins, while this protein complex antagonizes the ability of HNF-6 to recognize its target DNA sequence. Furthermore, we demonstrate that CCAAT/Enhancer Binding protein a(C/EBPalpha), but not the related C/EBPbeta protein, interacts with and provides transcriptional synergy with HNF-6 and that this protein complex leads to inhibition of C/EBPa target genes. The fact that the insulin-phosphatidylinositoi 3'-kinase (PI3K)-Akt signal transduction pathway inhibits HNF-6 transcriptional activation thus identifies a signaling pathway for regulating activity of these transcriptional synergies. Our studies suggest the hypothesis that interactions between liver transcription factors result in both transcriptional synergy and inhibition, thus providing a mechanism, which achieves diverse transcriptional function in response to transcription factor protein levels or activity. We will further characterize mechanisms of synergy and inhibition between hepatic transcription factors and investigate their regulation by the insulin-Akt pathway in vivo with the following four specific aims: 1) We will test the hypothesis that diminished HNF-6 transcriptional activity is due to Akt phosphorylation directly or indirectly through Akt inhibition of Glycogen Synthase Kinase 3 (GSK-3) activity. We will determine whether the Akt pathway negatively regulates HNF6-FoxA2 transcriptional synergy. 2) We will examine whether inhibiting HNF6-FoxA2 transcriptional synergy in vivo with a doxycycline inducible HNF6 dominant negative protein disrupts liver development and adult hepatocyte gene expression. 3) We will define the sequences involved in mediating liver-specific C/EBPa-HNF6 transcriptional synergy. We test the hypothesis that both the Akt pathway and increased C/EBPbeta levels negatively regulate C/EBPalpha-HNF6 transcriptional synergy. 4) We will use the Chromatin Immunoprecipitation (CHIP) assay to examine whether the HNF-6, FoxA2 and C/EBPalpha proteins form synergistic complexes with liver target promoters in vivo. Completion of these studies will provide a better understanding of the mechanisms involved in transcriptional synergy and inhibition among liver transcription factors, which mediate regulation of hepatocyte-specific gene expression.